The primary focus of this research project is to investigate the mechanisms through which apolipoprotein A-I (apo A-I) acts on high density lipoproteins (HDL) to activate lecithin:cholesterol acyltransferase (LCAT) thereby promoting the maturation of HDL particles and the removal of excess cholesterol from peripheral tissues of the body. The overall hypothesis we are testing is that negatively and positively charged residues within repeat 6 are responsible for LCAT activation either through direct apo A-I to LCAT interactions or through the formation a intra-molecular salt bridge(s) which stabilize a conformation of apo A-I that interacts with LCAT. To test this hypothesis, we will carry out three specific aims. The first aim will be to determine the lipid-bound conformation of apo A-I on discs and spheres using fluorescence resonance energy transfer (FRET) and chemical crosslinking combined with electrospray mass spectroscopy. This information will help to establish a "conformational map" of lipid-bound apo A-I before binding to LCAT and aid in determining if the conformation of lipid-bound apo A-I is altered once LCAT binds to its substrate. Our second aim will be to map contact sites between apo A-I and LCAT using a photoactivatible crosslinker attached to cysteine residues placed at specific sites within repeat 6. These studies will allow us to definitively show that apo A-I and LCAT interact during activation. These studies will also tell us specifically which apo A-I side-chain residue(s) potentially interact with LCAT. Thirdly, we will determine the in vitro and in vivo significance of the 4 negative and 6 positively charged residues within repeat 6. These studies will be carried out using apo A-I mutagenesis and in vitro LCAT assays. We will also develop and study transgenic animals expressing mutant forms of apo A-I. In summary, these studies will allow us to elucidate the mechanism responsible for activation of LCAT by apo A-I and the metabolic importance of apo A-I charged repeat 6 residues on HDL metabolism and the development of atherosclerosis. It is our hope that this information may eventually lead to new therapies for the prevention and treatment of atherosclerosis and CHD.